Three-Dimensional Microfabricated Bioreactors with Embedded Capillary Network

Abstract

In an aspect, the present invention uses projection micro stereolithography to generate three-dimensional microvessel networks that are capable of supporting and fostering growth of a cell population. For example, provided is a method of making a microvascularized bioreactor via layer-by-layer polymerization of a photocurable liquid composition with repeated patterns of illumination, wherein each layer corresponds to a layer of the desired microvessel network. The plurality of layers are assembled to make a microvascular network. Support structures having different etch rates than the structures that make up the network provides access to manufacturing arbitrary geometries that cannot be made by conventional methods. A cell population is introduced to the external wall of the network to obtain a microvascularized bioreactor. Provided are various methods and related bioreactors, wherein the network wall has a permeability to a biological material that varies within and along the network.

1 . A method of making a microvascularized bioreactor, said method comprising:
 a. providing a photocurable liquid composition having a top surface;   b. providing a source of light capable of curing at least a portion of said composition;   c. illuminating said composition top surface with said light, wherein said illumination is in a pattern thereby simultaneously generating a polymerized pattern layer having a layer thickness;   d. immersing said polymerized pattern layer into said composition depth by a vertical displacement corresponding to said layer thickness;   e. waiting a surface dwell time for said surface to become substantially level;   f. repeating said illuminating step to generate an adjacent polymerized pattern layer, wherein said repeating step is repeated for a number of steps to generate a microvascular network having an interior surface and an exterior surface; and   g. contacting said exterior surface with a cell population, thereby obtaining said microvascularized bioreactor;   wherein said microvascular network has a permeability to a biological material that varies with location within said network.   
     
     
         2 . The method of  claim 1  wherein said permeability is selected to optimize diffusion of said biological material through a network wall between said interior and exterior surfaces. 
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 2 , wherein said permeability varies along a longitudinal direction or a radial direction. 
     
     
         5 - 7 . (canceled) 
     
     
         8 . The method of  claim 1  further comprising:
 providing gray scale illumination in at least one illumination step, 
 wherein said gray scale illumination is provided by a gray scale mask having a plurality of pixels, each pixel capable of providing a plurality of grayscale shades each having a unique intensity. 
 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 8 , wherein said gray scale illumination has a minimum illumination intensity sufficient to cause polymerization, so that said gray scale illumination generates a polymerized layer with variable cross-linking, thereby providing variable permeability in said polymerized layer. 
     
     
         11 . The method of  claim 8 , wherein said gray scale illumination has a minimum intensity insufficient to generate polymerization, thereby providing a polymerized layer having features with different heights. 
     
     
         12 - 16 . (canceled) 
     
     
         17 . The method of  claim 1 , further comprising an electrowetting step to reduce surface dwell time, said electrowetting step comprising:
 a. providing a two-fluid interface operably connected to said top surface, wherein one fluid is conductive and the other fluid is non-conductive; and   b. applying a voltage to said conductive fluid to flatten said interface, thereby decreasing said surface dwell time.   
     
     
         18 . The method of  claim 17  wherein said two-fluid interface comprise said one fluid that is polyethylene glycol diacrylate (SPEGDA) conductive fluid on the bottom said other fluid is octane nonconductive fluid on top. 
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 1 , further comprising:
 a. obtaining an in silico image of an in vivo microvascular network, wherein said image comprises a plurality of layers; and   b. using each of said layers to generate each of said polymerized layers thereby making a microvascular network having a geometry corresponding to said in vivo microvascular network.   
     
     
         21 - 24 . (canceled) 
     
     
         25 . The method of  claim 1 , wherein said microvascular network has an upstream inlet port and a downstream outlet port, said method further comprising:
 a. introducing a culture media capable of sustaining said cell population to said inlet port at an inlet flow-rate; and   b. removing said culture media that has transited said microvascular network at said outlet port   wherein said cell population ation produces a compound that diffusues from said exterior surface to said interior surface, and is subsequently collected at said outlet port.   
     
     
         26 - 35 . (canceled) 
     
     
         36 . The method of  claim 1 , wherein said illuminating step comprises
 a. illuminating a first region with a first light exposure; and   b. illuminating a second region with a second light exposure, wherein said second light exposure has an intensity that is less than said first light exposure intensity, or said first second light exposure has a duration that is less than said first light exposure duration, or both;   thereby generating a polymer in said first region that has a cross-linking density that is greater than said second region cross-linking density.   
     
     
         37 . The method of  claim 36  further comprising selecting said first and second light exposure intensity, duration, or both to generate an etch rate for said first region polymer and said second region polymer when exposed to an etchant that is at least 10 times different from each other. 
     
     
         38 . (canceled) 
     
     
         39 . The method of  claim 36 , wherein said first region polymer is a microstructure that is a part of said microvascular network and said second region polymer is a sacrificial element, said method further comprising the step of contacting said sacrificial element with an etchant to at least partially remove said sacrificial element. 
     
     
         40 - 42 . (canceled) 
     
     
         43 . A method of making a three-dimensional device, said method comprising:
 a. providing photocurable liquid composition having a top surface;   b. providing a light source capable of curing at least a portion of said composition;   c. illuminating said composition top surface with gray scale illumination, wherein said gray scale illumination is a pattern of light intensity or duration that generates a pattern of polymer having a spatially varying cross-linking density; and   d. contacting said polymer with an etchant that selectively removes polymer having a lower cross-linking density;   thereby making a three-dimensional device.   
     
     
         44 . The method of  claim 43  further comprising making a plurality of polymer layers by:
 a. illuminating said composition top surface with said light source, wherein said illumination is in a pattern thereby simultaneously generating a polymerized pattern layer having a layer thickness; 
 b. immersing said polymerized pattern layer into said composition depth by a vertical displacement corresponding to said layer thickness; 
 c. waiting a surface dwell time for said surface to become substantially level; and 
 d. repeating said illuminating step to generate an adjacent polymerized pattern layer, wherein said repeating step is repeated for a number of steps to generate a three-dimensional structure having at least one element that is a sacrificial element having said lower cross-linking density that supports at least a portion of said three-dimensional structure during processing. 
 
     
     
         45 . The method of  claim 43 , further comprising selecting said gray scale illumination to generate a first region of polymer that is a sacrificial element and a second region of polymer that is a microstructure, wherein said sacrificial element provides physical support to said microstructure, wherein said sacrificial element has an etch rate that is selected from a range that is at least 5 to 10 times greater than said microstructure etch rate. 
     
     
         46 . (canceled) 
     
     
         47 . The method of  claim 45  wherein said microstructure comprises an overhang structure or a movable element. 
     
     
         48 . (canceled) 
     
     
         49 . A method of producing a biological material, said method comprising:
 a. providing a vascularized bioreactor having a three-dimensional network of microvessels capable of fostering a cell population, wherein said network has
 i. a polymeric wall with a lumen-facing side and a cell-facing side, wherein at least a portion of said wall is permeable to said biological material and said wall has a permeability that varies with microvessel position; 
 ii. an inlet port upstream of said network for introducing culture media to said network; 
 iii. an outlet port downstream of said network for removing said culture media; 
   b. obtaining an isolated cell population capable of producing said biological material or a precursor thereof;   c. contacting said cell population to at least a portion of said outward-facing side of said network wall;   d. culturing said cell population in said bioreactor by introducing a culture media to said inlet port to expose said network wall inward-facing side to said culture media; and   e. collecting said culture media that has transited said microvessel network at said outlet port;   wherein said cell population produces a biological material capable of diffusing from said cell population to said culture media via said network wall so that said collecting step collects at least a portion of said produced biological material, said biological material is a biofuel, pharmaceutical, drug, a prodrug, or any precursors thereof.   
     
     
         50 - 56 . (canceled) 
     
     
         57 . A vascularized bioreactor comprising:
 a. a three-dimensional network of microvessels having a wall made of a biocompatible polymer, wherein said wall has at least one parameter that varies with a longitudinal or a radial position within the network;   b. an inlet port for introducing culture media to said network; and   c. an exit port where culture media is removed from said network.   
     
     
         58 - 67 . (canceled) 
     
     
         68 . A method of calibrating a medical device comprising:
 a. providing a microvascular network made by the method of  claim 1  that supports a cell population;   b. introducing a challenge to said network;   c. imaging said microvascular network with a medical instrument to generate output data; and   d. calibrating said medical instrument with said output data.   
     
     
         69 - 71 . (canceled)